Investigating The Function Of Filamin B In Gene Transcription,Cell Proliferation And Migration

Filamin B(Filamin B-FLNB),encoded by the gene on chromosome 3,is a cytoskeletal protein that cross-linked with actin to form 3D cytoskeletal network and maintain cell morphology;it is involved in a variety of physiological and biochemical activities in cells,including transcriptional regulation and cell proliferation and so on.Recent studies have shown that FLNA and FLNB play key roles in tumorigenesis,skeletal disease,cell migration and adhesion.Our previous studies showed that FLNA inhibits gene transcription directed by Pol I and gene transcription by Pol ? in cell typespecific manner,subsequently represses cell proliferation.Since FLNB and FLNA belong to the filamin family,they have 80% homology in primary structure and co-localized in cytoplasm,in this project we sought to understand whether FLNB is also involved in gene transcription directed by Pol I and Pol III and affects the cell proliferation and migration.In order to achieve the above goal,we prepared the FLNB sh RNA lentivirus expression system that were used to infect 293 T and He La cells,the FLNB sh RNA stable expression cell line was selected.RT-q PCR was used to analyze Pol I and Pol III-directed gene transcription in these cell lines.The results showed that FLNB knockdown was able to significantly reduce RNA expression level,suggesting that FLNB is required for Pol I and Pol III-directed gene transcription.To confirm this finding,we performed similar experiments using 293 T and He La cells that were transfected FLNB-expressing vector,the results showed that FLNB overexpression significantly increased ribosomal RNAs,U6 sn RNA and 7SL RNA expression.These results indicate that FLNB is required to maintain normal gene transcription directed by Pol I and Pol III.Since FLNA knockdown promotes Pol I and Pol III gene transcription in cells,whether it abrogates the inhibition of gene expression caused by FLNB knockdown when FLNA expression is disrupted in FLNB knockdown cell lines.Using FLNB-FLNA double knockdown cell lines we performed RT-q PCR assays,the data showed that knockdown of FLNA up-regulated the expression of those genes affected by FLNB knockdown.This result suggests that FLNB and FLNA play an opposite role in the regulation of gene transcription by Pol I and Pol III.It has been shown that Pol I and Pol III gene transcriptions are correlated with cell proliferation,therefore,we tried to determine whether the change of FLNB expression level affect cell proliferation.The result from cell counting and MTT assays performed at different times showed that FLNB knockdown inhibited cell proliferation,while overexpression of FLNB enhanced cell proliferation.Whether FLNB regulates cell migration is not fully understood.Thus,we performed cell scratches and Transwell assays and analyze the effect of FLNB expression alteration on cell migration.The data showed that knockdown of FLNB inhibited cell migration,while FLNB overexpression promoted cell migration.Furthermore,the result from proliferation and migration assays using FLNA knockdown cell lines and FLNB-FLNA double knockdown cell lines demonstrated that FLNA and FLNB played an opposite role in the regulation of cell proliferation and migration;that is,FLNA inhibited cell proliferation and migration,while FLNB enhanced cell proliferation and migration.The proliferation rates of FLNB-FLNA knockdown cell lines was increased to a certain extent when compared to those of FLNB knockdown cell lines,however,the migration ability of double knockdown cell lines reduced.This result confirmed that FLNA played a positive role in cell migration when both FLNB and FLNA in 293 T and He La cells were knocked down,suggesting that the regulation of cell migration by FLNA depends on the expression of FLNB.Taken together,this study found that FLNB is essential to maintain normal Pol I and Pol III gene transcription,cell proliferation and cell migration,while the regulation of cell migration by FLNA depends on the presence or absence of FLNB in cells.These findings provide a basis for further exploring the mechanism of gene transcription and cell migration mediated by FLNB.